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Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence., Author summary Enteropathogenic Escherichia coli (EPEC) are pathogenic bacteria that cause infantile diarrhea. Upon ingestion, EPEC reaches the small intestine, where an injection device termed the type III secretion system is utilized to inject a set of effector proteins from the bacteria into the host cell. These proteins manipulate the localization and functions of host proteins, lipids and organelles and contribute to the emergence of the EPEC disease. The molecular mechanisms underlying the functions of the EPEC effector proteins are not completely understood. Here we show that early upon infection, two such effector proteins, EspF and Map, hijack host endosomes at bacterial adherence sites to facilitate endocytosis and recycling of plasma membrane proteins at these sites. The consequence of this event is the enrichment and mislocalization of host plasma membrane proteins at infection sites. One such protein is the transferrin receptor, which is a carrier for transferrin, whose function is to mediate cellular uptake of iron. Iron is a critical nutrient for bacterial growth and survival. We postulate that the unique manipulation of transferrin receptor endocytic membrane trafficking by EPEC plays an important role in its survival on the luminal surface of the intestinal epithelium.

Aim: To elucidate the interactions, uptake mechanisms and cytotoxicity profile of glucose-functionalized gold nanoparticles (2GF-GNPs), for expanding and advancing the recently proposed technology of metabolic-based cancer detection to a variety of cancer diseases. Methods: Several cell types with different metabolic features were used to assess the involvement of GLUT-1 and different endocytosis pathways in 2GF-GNP uptake, and the cytotoxicity profile of 2GF-GNPs. Results: Cellular uptake of 2GF-GNP strongly correlated with GLUT-1 surface expression, and occurred mainly through clathrin-mediated endocytosis. 2GF-GNPs showed no toxic effect on cell cycle and proliferation. Conclusion: These findings promote development of metabolic-based cancer detection technologies, and suggest that 2GF-GNPs may enable specific cancer detection in a wide range of tumors characterized by high GLUT-1 expression.

Detection of microscopic fission track (FT) star-shaped clusters, developed in a solid state nuclear track detector (SSNTD) by etching, created by fission fragments emitted from particles of fissile materials irradiated by neutrons, is a key technique in nuclear forensics and safeguards investigation. It involves scanning and imaging of a large area, typically 100–400 mm2, of a translucent SSNTD (e.g., polycarbonate sheet, mica, etc.) to identify the FT clusters, sparse as they may be, that must be distinguished from dirt and other artifacts present in the image. This task, if done manually, is time consuming, operator dependent, and prone to human errors. To solve this problem, an automated workflow has been developed for (a) scanning large area detectors, in order to acquire large images with adequate high resolution, and (b) processing the images with a scheme, implemented in ImageJ, to automatically detect the FT clusters. The scheme combines intensity-based segmentation approaches with a morphological algorithm capable of detecting and counting endpoints in putative FT clusters in order to reject non-FT artifacts. In this paper, the workflow is described, and very promising preliminary results are shown.

Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold nanoparticles tagged sample using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is then further enhanced also for tracking of single fluorescent particles within a three dimensional (3D) cellular environment based on image processing algorithms that can significantly increases localization accuracy of the 3D point spread function in respect to regular Gaussian fitting. All proposed concepts are validated both on simulated data as well as experimentally.

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Objectives The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action. Methods A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy. Results Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8. Conclusions We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus. We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited., Summary: A detailed analysis of posterior segment addition in an insect reveals that the growth zone is divided into two functional domains responsible for growth and differentiation.

This work presents a technique for a full 3D imaging of biological samples tagged with gold-nanoparticles (GNPs) using only two images, rather than many images per volume as is currently needed for 3D optical sectioning microscopy. The proposed approach is based on the Gerchberg-Saxton (GS) phase retrieval algorithm. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. In addition, since the method requires the capturing of two images only, it can be suitable for 3D live cell imaging. The proposed concept is presented and validated both on simulated data as well as experimentally.

The ability to track single fluorescent particles within a three dimensional (3D) cellular environment can provide valuable insights into cellular processes. In this paper, we present a modified nonlinear image decomposition technique called K-factor that reshapes the 3D point spread function (PSF) of an XYZ image stack into a narrow Gaussian profile. The method increases localization accuracy by ~60% with compare to regular Gaussian fitting, and improves minimal resolvable distance between overlapping PSFs by ~50%. The algorithm was tested both on simulated data and experimentally. This work presets a novel use of the nonlinear image decomposition technique called K-factor that reshapes the three dimensional (3D) point spread function (PSF) of an XYZ image stack into a narrow Gaussian profile. The experimentally obtained PSF of a Z-stack raw data that is acquired by a widefield microscope has a more elaborate shape that is given by the Gibson and Lanni model. This shape increases the computational complexity associated with the localization routine, when used in localization microscopy techniques. Furthermore, due to its nature, this PSF spreads over a larger volume, making the problem of overlapping emitters detection more pronounced. The ability to use Gaussian fitting with high accuracy on 3D data can facilitate the computational complexity, hence reduce the processing time required for the generation of the 3D superresolved image. In addition it allows the detection of overlapping PSFs and reduces the effects of the penetration of out of focus PSFs into in focused PSFs, therefore enables the increase in the activated fluorophore density by ~50%. The algorithm was tested both on simulated data and experimentally, where it yielded an increase in the localization accuracy by ~60% with compare to regular Gaussian fitting, and improved the minimal resolvable distance between overlapping PSFs by ~50%, making it extremely applicable to the field of 3D biomedical imaging

Multi-drug resistance (MDR) is still a major cause of the eventual failure of chemotherapy in cancer treatment. Different approaches have been taken to render these cells drug sensitive. Here, we attempted sensitizing drug-resistant cells from within, using a translocating immune conjugate approach. To that effect, a monoclonal antibody, C219, directed against the intracellular ATP-binding site of the membrane-anchored MDR transporter ABCB1 [P-glycoprotein (P-gp), MDR1], was conjugated to human immunodeficiency virus [HIV(37-72)Tat] translocator peptide through a disulfide bridge. Fluorescence-labelled IgG-Tat conjugates accumulated in drug resistant Chinese hamster ovary (CHO) cells within less than 20 min. Preincubation with C219-S-S-(37-72)Tat conjugate augmented calcein accumulation in drug-resistant CHO and mouse lymphoma cells, indicating reduction in ABCB1 transporter activity. A thioether conjugate C219-S-(37-72)Tat was ineffective, as were disulfide and thioether conjugates of an irrelevant antibody. Furthermore, in the presence of C219-S-S-(37-72)Tat, drug resistant cells were sensitized to colchicine and doxorubicin. Taken together, these findings demonstrate, as proof of principle, a novel approach for the reversal of MDR from within cells, by delivery of translocating immune conjugates as sensitizing agents towards chemotherapy.

In this work, a novel algorithm for improvement of blurred SEM and TEM images of metal ultra-thin films' microstructure has been developed. The algorithm, differently from other existing methods which are based on a priori knowledge about the inspected sample or the imaging system, takes into account the a priori knowledge about the microstructures grain size and shape ranges. The feasibility of the algorithm was examined on totally blurred HRSEM images of copper and silver films microstructures. Our study shows that this algorithm can also be used to improve indistinct images of filled trenches/vias. Thereby, the algorithm may obviate the need for time-consuming selection of SEM/TEM measurement conditions.

4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4'-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4'-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.

Herein we present a new application of a recently demonstrated fully synthetic "phage-like" system for screening of combinatorial mixtures in a live cell assay. The new application includes the direct synthesis of peptides and combinatorial libraries on 2 ∝m cross-linked mono-dispersed microspheres bearing a panel of fluorescence tags. Their characterization using classical chemical analysis as well as biological recognition of the synthesized sequences on the microspheres by specific antibodies, demonstrate the robustness of the system. Two biased positional combinatorial peptide libraries derived from peptide DUP-1 were synthesized and screened for affinity to prostate cancer PC-3 cell line as compared to DUP-1, a peptide with good affinity for this cell line. The best sub-library was deconvoluted and mixtures of deconvoluted peptides on microspheres were screened for identifying the sequences with the best affinity for cells. The best peptide, DUP-1 (1-12) 2 Ala with high affinity for PC-3 cells, was individually synthesized and validated in an independent binding assay. Finally, the cellular fate of DUP-1 (1-12) 2 Ala in PC-3 cell line is demonstrated using confocal laser scanning microscopy. Overall, these results demonstrate that the synthetic phage-like system recently assessed for screening mixtures of small organic molecules, can be also used for synthesis and screening of combinatorial libraries of peptides in live cell assays.

A new approach aimed to achieve microscopic imaging without objective lenses and based on wavelength multiplexing of the spatial object information is presented. The proposed method is used to develop, construct and experimentally validate a new type of optical microscope having no objective lens and no numerical reconstruction algorithms to allow imaging process. In order to extract the collected spatial information we use a spectrometer as part of our microscope system. Preliminary results are presented while considering two different types of one-dimensional (1-D) objects.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P2and PI(3,4,5)P3in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P2accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P2depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P2contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P3clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P3beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.

Background: In Alzheimer’s disease (AD), brain butyrylcholinesterase (BChE) co-localizes with β-amyloid (Aβ) fibrils. Aims: In vitro testing of the significance of this phenomenon to AD progress. Methods: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Aβ fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. Results: Both proteins at 1:50 and 1:25 ratios to Aβ dose-dependently prolonged the ThT lag time and reduced the apparent rate of Aβ fibril formation compared to Aβ alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Aβ alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Aβ prevented the formation of larger, over 150-nm-long, Aβ fibrils and reduced fibril branching compared to Aβ alone as quantified by macro programming of Image Pro® Plus software. Conclusion: Our findings demonstrate that rBChE interacts with Aβ fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-β-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.

Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume and is therefore time consuming and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold-particle tagged samples using only two images. The reconstructed field is free space propagated to all other focus planes using post processing and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is presented and validated both on simulated data as well as experimentally.

Nanocrystals of binary metal tellurides, such as PbTe, Ni2Te3, and Cu7Te5, are synthesized by the polyol method under microwave radiation and studied using X-ray powder diffraction, transmission electron microscopy, high-resolution transmission electron microscopy, and photoacoustic spectroscopy. Binary tellurides are formed only in the case when ethylene glycol is used as a reducing agent and only when the reaction is carried out under microwave radiation. No product is formed when the reactions are performed using a conventional technique of heating. The synthesis of the aforementioned metal tellurides is described, and the proposed mechanism of the reaction is discussed.

Fluorescence photobleaching was measured in dye-impregnated sol–gel/polymer composite glasses. These fluorescent glasses were used as the gain medium in a transverse-pumped solid-state dye laser. In this configuration, the fluorescent glass was excited by a pulsed Nd:YAG laser (about 6 mJ/pulse) either while placed in an optical cavity (i.e., functioning as a pulsed laser) or with the optical cavity blocked, so that lasing did not occur. The decay of the fluorescence signal versus cumulative excitation energy was recorded. We found that the rate of photobleaching decreased when the glass was lasing, as compared to the case where the optical cavity was blocked. This paper presents these results, and suggests a simple kinetic model that may explain this phenomenon.

We have previously described two nucleolar proteins, named p14 and p21, in MMTV-induced T cell lymphomas. These proteins were identified by a monoclonal antibody (M-66) generated from a nontumorigenic, immunogenic variant of S49 T cell lymphoma. While p14 was common to several MMTV-derived T cell lymphomas, p21 was found only in highly tumorigenic variants of S49 cells. Here we report that p14 is the leader peptide of the MMTV env precursor. The epitope recognized by M-66 contains a putative nuclear localization signal. Actinomycin D was found to induce redistribution of p14/p21 from the nucleolus to the nucleoplasm. p14 coimmunoprecipitated and colocalized with the cellular protein, B23. Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (HTLV).

The Zintl compounds Na 4 SnSe 4 , K 4 Sn 2 Se 6 , and K 4 Sn 3 Se 8 were prepared for the first time using the microwave-assisted solid-state reaction method. The heteroanions of these phases represent oligomeric group of (zero-dimensional) molecular anions: [SnSe 4 ] 4− , [Sn 2 Se 6 ] 4− , [Sn 3 Se 8 ] 4− . The compounds were characterized using Powder XRD, SEM-EDS, and their electronic properties were studied using photoacoustic spectroscopy. The microwave method was found to be simple, fast and efficient for the preparation of these Zintl compounds.

Nanocrystals of Ag2E (E = Se, Te) were prepared using the polyol reduction method under sonochemical irradiation. Particles were characterized using TEM, HRTEM, SEM, and XRD. The optical properties of the products were studied using photoacoustic spectroscopy (PAS). The differences between the as-prepared samples and single crystals and method of the nanoparticle formation are discussed.

Nanoparticles of the Cd1 − xZnxSe alloy with different stoichiometries (x = 0.1, 0.2, 0.3) have been prepared using the microwave-assisted polyol method. In this simple and quick reaction the polyol (ethylene glycol) is both the solvent and the reducing agent. XRD studies show that nanoparticles are formed in the wurtzite (hexagonal) form with an average diameter of the nanocrystals of 5–8 nm. Electron microscopy studies show that these particles form monodisperse spherical aggregates with a diameter of approximately 200 nm. Electronic properties of the as-prepared alloys have been studied using photoacoustic spectroscopy (PAS). Also, the particles have been characterized using TGA, DSC, XPS and EDS analysis. The results of these studies are presented in this paper.

Biocompatible hydrogel nanoparticles are prepared by polymerization and cross-linking of N-isopropyl acrylamide in a micelle template formed by block copolymers macro-monomers at high temperature. Different monomer ratios form, at high temperature, well-defined micelles of different sizes which are further polymerized leading to nanoparticles with varied sizes from 20 to 390 nm. Physico-chemical characterization of the nanoparticles demonstrates their composition and homogeneity. The NPs were tested in vitro and in vivo biocompatibility assays, and their lack of toxicity was proven. The NPs can be labeled with fluorescent probes, and their intracellular fate can be visualized and quantified using confocal microscopy. Their uptake by live stem cells and distribution in whole developing animals is reported. On the basis of our results, a mechanism of nanoparticle formation is suggested. The lack of toxicity makes these nanoparticles especially attractive for biological applications such as screening and bio-sensing.

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

A photoacoustic spectrometer is described which has been constructed to measure the spectra of photovoltaic or other solid materials in the ultraviolet (UV), visible and near infrared regions. The range of this photoacoustic spectrometer is from 0.3 to 1.8 μm. The spectrometer was used to characterize the optical properties of polycrystalline and single phase p-CuInSe2 thin films which were grown by selenization of sputtered Cu/In multilayer at a temperature of 500°C. The films showed a three-fold optical structure with three high energy transitions, which is the characteristic behavior of the material. The photoacoustic spectrum is reported and discussed.

Porous glasses were prepared using the sol–gel method. The mix of catalysts used during preparation was changed in order to produce two glasses with two different densities. Fluorescent dyes were incorporated into these glasses by diffusion into the glass. The resulting fluorescent glasses were characterized using confocal microscopy, which allows thin optical sections to be imaged at up to 200 μm below the sample surface. In addition, fluorescence recovery after photobleaching (FRAP) was used to demonstrate diffusion of the dye within wet samples. The results demonstrate the potential for non-invasive optical characterization of fluorescent sol–gel glasses, and in particular local measurement of diffusion coefficients within the glass matrix.

The microwave-assisted polyol reduction method was applied to the preparation of CdSe “nanoballs”. The cubic and hexagonal forms of the CdSe nanoparticles obtained were analyzed by XRD, TEM, HRTEM, UV-VIS, photoluminescence (PL) and photoacoustic spectroscopy. Formation of the hexagonal or cubic form of CdSe depends on the glycol used for reaction. It was found that “nanoballs” are built-up of clusters of CdSe with sizes of a few nanometers. A strong blue shift is observed in the absorption spectrum, as expected for size quantization. A possible reaction mechanism is provided.

The compartments involved in polarized exocytosis of membrane proteins are not well defined. In this study we hypothesized that newly synthesized polymeric immunoglobulin receptors are targeted from the trans-Golgi network to endosomes prior to their appearance on the basolateral cell surface of polarized Madin-Darby canine kidney cells. To examine this hypothesis, we have used an assay designed to measure the meeting of newly synthesized receptors with a selective population of apical or basolateral endosomes loaded with horseradish peroxidase. We found that in the course of basolateral exocytosis, the wild-type polymeric immunoglobulin receptor is targeted from the trans-Golgi network to apical and basolateral endosomes. Phosphorylation of a Ser residue in the cytoplasmic tail of the receptor is implicated in this process. The biosynthetic pathway of apically sorted polymeric immunoglobulin receptor mutants similarly traversed apical endosomes, raising the possibility that apical receptors are segregated from basolateral receptors in apical endosomes. The post-endocytic pathway of transcytosing and recycling receptors also passed through apical endosomes. Together, these observations are consistent with the possibility that the biosynthetic and endocytic routes merge into endosomes and justify a model suggesting that endosomal recycling processes govern polarized trafficking of proteins traveling in both pathways.

Luminescence from chromium–neodymium-doped lithium niobate (LiNbO3) was experimentally measured to determine the degree of non-radiative energy transfer between chromium and neodymium ions. Evidence is presented for two different time constants for emission from chromium ions in the material, indicating that non-radiative transfer does occur. Differences between quasi-continuous pumping and pulsed excitation are discussed.

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo , and lamins bind specifically to chromatin in vitro . Deletion mutants of Drosophila lamin Dm 0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm 0 tail domain. The apparent K d of binding of the lamin Dm 0 tail domain was found to be approximately 1 μM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm 0 . Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm 0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm 0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm 0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm 0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo .

We provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled gamma-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. gamma-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. gamma-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of gamma-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from the trans-Golgi network.

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

This paper presents results based on concentration quenching of fluorescence polarization (cqfp), which demonstrate the feasibility of using cqfp to measure changes in hydrogel-membrane hydration. This is accomplished by binding fluorescent molecules to the hydrogel matrix, and showing that the fluorescence polarization is a monotonic function of fluorophore concentration. Films based on crosslinked 2-hydroxyethyl methacrylate containing lissamine side groups (10−4-10−7 mol/l) were mounted in a special cell which provided an aqueous environment, and in which polarized fluorescence could be measured. An argon-ion laser provided polarized excitation at 488nm, and the polarized fluorescent emission was detected. The correlation between the fluorescence polarization and the bound dye concentration was found to correspond to the theoretically expected behavior.

Planar waveguides made of titania and organically modified silica (osmosil) and doped by laser dyes were prepared using the sol-gel method. The film structure and morphology were studied using absorption spectroscopy in the UV and by atomic force microscopy (AFM). The size of the titania nanoparticles was determined from the increase in the energy gap between the valence and conduction bands calculated from the UV absorption spectra. The films' morphology was studied from the AFM images. The doped dye molecules are in the organic phase of the hybrid glass, the fluorescence yield and photostability increase with the oemosil content of the glass; those trends are connected to the decrease in the film pore size.

Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.

A room temperature sonochemical method for the preparation of GaSb nanoparticles using less hazardous Ga and antimony chloride (SbCl(3)) as the precursors has been described. The formation of GaSb has been confirmed by means of XRD, EDAX, and XPS characterization. TEM and SAED results show that the as-prepared solid consists of nanosized GaSb crystals with sizes in the range 20-30 nm. The photoacoustic spectrum result reveals that the GaSb nanoparticles have a direct band gap of about 1.21 eV. On the basis of the control experiments and the extreme conditions produced by ultrasound, an ultrasound-assisted in-situ reduction/combination mechanism has been proposed to explain the reaction.

In this paper, we characterize the dynamic nature of the full amyloid beta (1–40) (Aβ (1–40)) aggregates. We labeled the peptide with either 5-carboxytetramethylrhodamine (TAMRA) or with fluorescein-isothiocyanate (FITC). The labeled peptides were mixed after separate fibrillization, and the dynamic changes in the structure of the fibrils were imaged using confocal microscopy. Fluorescence resonance energy transfer (FRET) measurements showed that the Aβ (1–40) peptides detach from and reattach to the fibrils in a biologically relevant timescale (days). With time, the two peptides mix at the molecular level. This process is concentration dependent and occurs primarily in the external parts of the aggregates with a half time between 4 and 7 days. This study shows that the combination of confocal microscopy and FRET analysis is a facile method for studying dynamic processes in supra-molecular aggregates.

A room temperature sonochemical method for the preparation of GaSb nanoparticles using less hazardous Ga and antimony chloride (SbCl3) as the precursors has been described. The formation of GaSb has been confirmed by means of XRD, EDAX, and XPS characterization. TEM and SAED results show that the as-prepared solid consists of nanosized GaSb crystals with sizes in the range 20−30 nm. The photoacoustic spectrum result reveals that the GaSb nanoparticles have a direct band gap of about 1.21 eV. On the basis of the control experiments and the extreme conditions produced by ultrasound, an ultrasound-assisted in-situ reduction/combination mechanism has been proposed to explain the reaction.

Nanocrystals of Ag2E (E = Se, Te) were prepared using the polyol reduction method under sonochemical irradiation. Particles were characterized using TEM, HRTEM, SEM, and XRD. The optical properties of the products were studied using photoacoustic spectroscopy (PAS). The differences between the as-prepared samples and single crystals and method of the nanoparticle formation are discussed.

A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.

Multiplexed imaging and illumination have been used to recover enhanced arrays of intensity or spectral reflectance samples, per pixel. However, these arrays are often not the ultimate goal of a system, since the intensity is a result of underlying object characteristics, which interest the user. For example, spectral reflectance, emission or absorption distributions stem from an underlying mixture of materials. Therefore, systems try to infer concentrations of these underlying mixed components. Thus, computational analysis does not end with recovery of intensity (or equivalent) arrays. Inversion of mixtures, termed unmixing, is central to many problems. We incorporate the mixing/unmixing process explicitly into the optimization of multiplexing codes. This way, optimal recovery of the underlying components (materials) is directly sought. Without this integrated approach, multiplexing can even degrade the unmixing result. Moreover, by directly defining the goal of data acquisition to be recovery of components (materials) rather than of intensity arrays, the acquisition becomes more efficient. This yields significant generalizations of multiplexing theory. We apply this approach to fluorescence imaging.

Measurement of the time-response of the excited-state concentration of manganese has confirmed our previous hypothesis of fast energy transfer between manganese and thulium in fluoride glasses.